Cloning And Co-expression Of D-arabitol Dehydrogenase And Xylitol Dehydrogenase Genes From Gluconobacter Thailandicus

Due to its high sweetness, low calories and being not affected by insulin metabolism features,xylitol received much attention and widely used in food, medicine and chemical industry areas. In recent years, as green and sustainable development, the environmental protection consciousness was enhanced. At the same time, biological production of xylitol is becoming a hot global research topic. D-arabitol dehydrogenase(ArDH) and xylitol dehydrogenase(XDH) are two critical and speed-limited enzymes in the xylitol biosynthetic pathway. Their high-efficiency expression plays a vital role in the biotransformation of D-arabitol into xylitol. So the construction of xylitol engineering bacteria which contains high-performance ArDH and XDH genes is of great significance to improve the production rate and realize industrial production.In this study, the ardh gene encoding ArDH was cloned from Gluconobacter thailandicus. Then recombinant plasmid pET28a-ardh was constructed using expressing vector p ET28 a. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) analysis revealed that the recombinase has the predicted molecular weight about 27 kDa. The enzyme was purified by nickel-chelate chromatography combined with Sephacryl S-300 H gel filtration and biochemically characterized. With D-xylulose as the substrate, the enzyme had maximum reduction activity at pH value 5.0 and temperature 30℃, and specific activity was11.46U/mg. The apparent Km and Vmax values of the enzyme for D-xylulose were 27mmol/L and 22.3μmol/min/mg, respectively. Result showed that the recombinase had maximum oxidation activity at pH value 9.0 and temperature 30℃ when using D-arabitol as the substrate. And the specific activity of recombinase was 27.72U/mg. The apparent Km and Vmax values of the enzyme for D-arabitol are 12.5mmol/L and 52.8μmol/min/mg, respectively.In addition,the xdh gene encoding XDH was also cloned from G.thailandicus, Then recombinant plasmid pET28a-xdh was constructed using expressing vector pET28 a. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) analysis revealed that the the recombinase of recombinant BL21(DE3)-xdh has the predicted molecular weight about 28 kDa. The enzyme was purified by nickel-chelate chromatography combined with Sephacryl S-300 H gel filtration and biochemically characterized. With D-xylulose as the substrate, the enzyme had maximum reduction activity at pH value 5.0 and temperature 30℃, and specific activity was 126.3U/mg. The apparent Km and Vmax values of the enzyme for D-xylulose were 11.2mmol/L and 134.2μmol/min/mg, respectively. Result showed that the recombinase had maximum oxidation activity at pH value 11.0 and temperature 35℃ when using xylitol as the substrate. And the specific activity of recombinase was 28.6U/mg. The apparent Km and Vmax values of the enzyme for xylitol were 78.6mmol/L and 53.6μmol/min/mg, respectively.Furthermore, recombinant strain contained vector pET28a-xdh-ardh was also constructed based on the recombined pET28a-xdh and pET28a-ardh. Shaking flask fermentation was conducted for recombinant stain BL21(DE3)-xdh-ardh and G. thailandicus respectively. The results showed that the recombinant strain producted 23.7 g/L xylitol from 80 g/L D-arabitol with the onversion rate of 29.6%, whereas G. thailandicus produced 12.9 g/L xylitol from 80 g/L D-arabitol with the conversion rate of 29.6%, so the onversion rate of recombinant strain was improved.